RESUMEN
Lysophosphatidic acid (LPA) is an extracellular signaling mediator with a broad range of cellular responses. Three G-protein-coupled receptors (Edg-2, -4, and -7) have been identified as receptors for LPA. In this study, the ectophosphatase lipid phosphate phosphatase 1 (LPP1) has been shown to down-regulate LPA-mediated mitogenesis. Furthermore, using degradation-resistant phosphonate analogs of LPA and stereoselective agonists of the Edg receptors we have demonstrated that the mitogenic and platelet aggregation responses to LPA are independent of Edg-2, -4, and -7. Specifically, we found that LPA degradation is insufficient to account for the decrease in LPA potency in mitogenic assays, and the stereoselectivity observed at the Edg receptors is not reflected in mitogenesis. Additionally, RH7777 cells, which are devoid of Edg-2, -4, and -7 receptor mRNA, have a mitogenic response to LPA and LPA analogs. Finally, we have determined that the ligand selectivity of the platelet aggregation response is consistent with that of mitogenesis, but not with Edg-2, -4, and -7.
Asunto(s)
Lisofosfolípidos/farmacología , Fosfatidato Fosfatasa/fisiología , Receptores de Superficie Celular/fisiología , Receptores Acoplados a Proteínas G , Línea Celular , ADN/biosíntesis , Relación Dosis-Respuesta a Droga , Humanos , Modelos Biológicos , Fosfatidato Fosfatasa/antagonistas & inhibidores , Agregación Plaquetaria , Receptores de Superficie Celular/agonistas , Receptores del Ácido LisofosfatídicoRESUMEN
The cysteinyl leukotrienes-leukotriene C4(LTC4), leukotriene D4(LTD4) and leukotriene E4(LTE4)-are important mediators of human bronchial asthma. Pharmacological studies have determined that cysteinyl leukotrienes activate at least two receptors, designated CysLT1 and CysLT2. The CysLT1-selective antagonists, such as montelukast (Singulair), zafirlukast (Accolate) and pranlukast (Onon), are important in the treatment of asthma. Previous biochemical characterization of CysLT1 antagonists and the CysLT1 receptor has been in membrane preparations from tissues enriched for this receptor. Here we report the molecular and pharmacological characterization of the cloned human CysLT1 receptor. We describe the functional activation (calcium mobilization) of this receptor by LTD4 and LTC4, and competition for radiolabelled LTD4 binding to this receptor by the cysteinyl leukotrienes and three structurally distinct classes of CysLT1-receptor antagonists. We detected CysLT1-receptor messenger RNA in spleen, peripheral blood leukocytes and lung. In normal human lung, expression of the CysLT1-receptor mRNA was confined to smooth muscle cells and tissue macrophages. Finally, we mapped the human CysLT1-receptor gene to the X chromosome.
Asunto(s)
Proteínas de la Membrana , Receptores de Leucotrienos/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Células COS , Mapeo Cromosómico , Clonación Molecular , Humanos , Antagonistas de Leucotrieno , Leucotrieno D4 , Pulmón/metabolismo , Macrófagos Alveolares/metabolismo , Datos de Secuencia Molecular , Músculo Liso/metabolismo , Receptores de Leucotrienos/química , Receptores de Leucotrienos/genética , Distribución Tisular , Transfección , Cromosoma X , Xenopus laevisRESUMEN
Lysophosphatidic acid (LPA) has associated with it an intriguing cell biology that is thought to be mediated through its interaction with G-protein coupled receptor(s). In an effort to extend the structure-activity relationships of LPA, we have produced a series of LPA analogues in which the glycerol core in LPA was replaced with conformationally restricted aryl substructures. The aryl substructures encompassed aminophenol, resorcinol, dihydroxy benzophenone, and tocopherol systems. The benzophenone moiety was investigated both as a conformationally restricting substructure for LPA and as a possible photoreactive alkylating agent for the LPA receptor(s). All LPA analogues were evaluated for their potency and efficacy in mobilizing calcium ions from internal stores in MDA MB-231 cells. Ten of the 14 analogues exhibited activity in this assay at doses up to 5 microM; none of the compounds exhibited nonreceptor-mediated lytic activity at this maximal concentration. The receptor response showed surprising tolerance for manipulation in the backbone region of LPA, although none of the compounds were equipotent to LPA. This tolerance for a variety of structures has given us new leads into the realization of novel agonists and antagonists of the LPA receptor(s).
Asunto(s)
Lisofosfolípidos/síntesis química , Receptores Acoplados a Proteínas G , Calcio/metabolismo , Humanos , Lisofosfolípidos/química , Lisofosfolípidos/farmacología , Conformación Molecular , Imitación Molecular , Receptores de Superficie Celular/efectos de los fármacos , Receptores del Ácido Lisofosfatídico , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Two human isoforms of membrane associated phosphatidic acid phosphatase have been described (PAP-2a and -2b), and both enzymes have been shown to have broad substrate specificity and wide tissue distribution [Kai et al., J. Biol. Chem. 272 (1997) 24572-24578]. With this report we describe a third isoform, PAP-2c, that we found by searching the database of expressed sequence tags (dbEST) with PAP-2a and PAP-2b sequences. Key structural features described previously in PAP-2a and -2b, including the glycosylation site, putative transmembrane domains, and the proposed catalytic site, are conserved in the novel phosphatase. The kinetics of the three enzymes were compared using as substrates phosphatidic acid, lysophosphatidic acid, and N-oleoyl ethanolamine phosphatidic acid. Km values for each of the substrates, respectively, were (in microM) PAP-2a: 98, 170, 116; PAP-2b: 100, 110, 56; and PAP-2c: 150, 340, 138. Expression of PAP-2c mRNA is more restricted than the two previously described isoforms.
Asunto(s)
Isoenzimas/metabolismo , Fosfatidato Fosfatasa/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN Complementario/genética , Humanos , Isoenzimas/genética , Cinética , Lisofosfolípidos/farmacología , Datos de Secuencia Molecular , Fosfatidato Fosfatasa/genética , Ácidos Fosfatidicos/metabolismo , ARN Mensajero/análisis , Proteínas Recombinantes , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Linfocitos TRESUMEN
Despite an intriguing cell biology and the suggestion of a role in pathophysiological responses, the mechanism of action of such lipid phosphoric acid mediators as lysophosphatidic acid (LPA) remains obscure, in part because of an underdeveloped medicinal chemistry. We report now the agonist activity of a synthetic phospholipid in which the glycerol backbone of LPA is replaced by L-serine. Like LPA, the L-serine-based lipid mobilizes calcium and inhibits activation of adenylyl cyclase in the human breast cancer cell line MDA MB231. Treatment with LPA desensitizes MDA MB231 cells to subsequent application of the L-serine compound; when the order of application is reversed, however, the L-serine compound does not prevent calcium mobilization by LPA, which might indicate the existence of two LPA receptors in these cells. The analogous D-serine-based phospholipid was distinctly less potent than the L-isomer in those assays; this finding demonstrates stereoselectivity by an LPA receptor. Unlike LPA, the L-serine-based lipid does not evoke a chloride conductance in Xenopus laevis oocytes, but injection of poly(A)+ RNA from HEK 293 cells confers this phenotype on the oocyte. The latter result has practical importance in that it allows use of the frog oocyte for expression cloning of an LPA receptor DNA, an assay system made problematic by the oocyte's strong endogenous response to LPA.